From the sequence, it might be possible to __, and possibly to __
Sequencing millions of base pairs in one shot is impossible right now. In fact, the max amount of sequence that can be read in a single experiment is __.
identify the genes present in the DNA molecule
deduce the functions of those genes
How is this sequencing performed?
This is done by breaking the long DNA molecules down into many short fragments, each of which is individually sequenced. The sequence is then assembled by searching for overlaps.
Automated techniques are made to make this easier, with the sequencing experiments carried out by robots, and computer programs that find the __and build up the __
- contiguous blocks of sequences, or contigs
The steps in DNA sequencing: Step One
Prepare a collection of short DNA fragments ready for sequencing, which is done by DNA cloning; the resulting collection is called a clone library
How do you prepare a clone library? Step ONe
First, break down the DNA fragments by sonication, a technique that uses high-frequency sound waves to make random cuts in the molecules
How do you prepare a clone library? Step Two
Next, separate the fragments resulting from sonication so that each one can be sequenced individually
What does separating the fragments require?
This requires a vector, a DNA molecule that is able to replicate inside a cell of the bacterium coli and usually a modified version of a naturally occurring, self-replicating DNA molecule called a plasmid.
Explain the plasmid in its replicative state
- In its replicative state, the plasmid is a circular molecule, but at the start of the cloning experiment it is obtained in a linear form
- The linear vector molecules are mixed with the DNA fragments, and a DNA ligase enzyme is added, which joins DNA molecules together end-to-end
- This produces a recombinant vector
How do you prepare a clone library? Step Three
In the next stage, the molecules resulting from ligation are mixed with E. coli cells, which have been chemically treated so that they are competent, a term used to describe cells that are able to take up DNA molecules from their environment
what happens after exposre of E coli cells with the plasmid?
Some molecules are taken up by bacteria—but usually no more than one Linear molecules, and circular molecules that lack the vector sequences, cannot be propagated inside a bacterium.
How do you prepare a clone library? Step Four
The bacteria are then spread onto a solid agar medium to divide and produce colonies, each containing copies of the vector This is the clone library
The chain termination method
sequence obtained by making copies of a cloned DNA fragment in a way that enables the position of each of the four nucleotides to be identified; it makes use of a purified DNA polymerase
Explain the method of chain termination
- First, the recombinant vector molecules are purified from one of the clones in the clone library.
- A short piece of DNA called an oligonucleotide is attached to the vector part of the recombinant DNA molecule, just adjacent to the site into which the cloned fragment has been inserted.
- This oligonucleotide acts as a primer for synthesis of the new strand
- To sequence it, dideoxynucleotides are used, which terminate the elongation process
ddNTPs lack an –OH group at the 3’ position This produces fragments of all different lengths, which are then run on a capillary gel electrophoresis and read from the bottom up
What does the migration rate of eac molecule through the capillary gel depend on?
- its electric charge
- Each phosphodiester bond in a DNA molecule carries a single negative electric charge, so the overall negative charge of the polynucleotide is directly proportional to its length
To read the sequence better, what has been done?
To read it more easily, the four dideoxynucleotides emit fluorescent signals of different wavelengths, which allow each to be identified by reading the different colors
The chain termination method allows what?
This method allows a maximum of 750 bp to be sequenced at a time